The process of somatic hypermutation (SHM) of immunoglobulin genes is initiated by the cytidine deaminase AID creating cytidine (C) to uridine (U) transition mutations. Outside of SHM, U is faithfully repaired to C with the help of base excision repair (BER) and mismatch repair (MMR). During SHM, paradoxically, these repair mechanisms are recruited together with translesion DNA polymerases to create mutations at and near the U by error-prone repair. A critical hallmark of SHM is the restriction of the mutations to the 5'end of Ig genes, including the V(D)J region, but sparing the constant region. In this way, an enormous diversity of antigen-binding variable regions is created while protecting the biological functions of the antibody constant regions. We have considered two ways in which this V-region restriction may come about. First, AID may only be active in the 5'region, and second, AID may act throughout the gene but error-prone repair would only operate in the 5'region. In extensive experiments with mice that are defective in BER or MMR or both, we found the same pattern of mutations as in wild type mice, namely restriction to the variable region and its immediate flanks. This leads to the conclusion that AID does not act at the 3'portion of the Ig gene. We now wish to determine how exactly AID interacts with the sequences in which it deaminates Cs. Based on experiments where the constant region was mutated when the Ig gene promoter was duplicated in front of the constant region, we proposed the following model: AID associates with the transcription complex near the promoter, travels with the RNA polymerase during transcript elongation, deaminates Cs in single- stranded DNA arising in negative DNA supercoils behind the polymerase, and dissociates from the transcription complex within ~2kbp from the promoter due to limited affinity. We propose a novel and untried approach to test this model. The experiments will be carried out with a modified chicken B cell line, DT40 PseudoV-del, that suffers cytydine deaminations and subsequent SHM at a high rate. The modified line was produced by J.M. Buerstedde by deleting the pseudo V genes of the parent DT40 line so that the modified cells cannot carry out Ig gene conversion. In response to AID, the modified pseudoV-del DT40 cells undergo only SHM and do so at a high rate since AID- induced uridines are not directed toward gene conversion. They are therefore an ideal tool to study SHM. If the results of these exploratory experiments are clear, either supporting or rejecting the model of SHM described above, they will provide a platform and methods to unravel remaining questions, such as how AID is targeted only to Ig and some other genes, including some of known relevance to cancer and other diseases. PUBLIC HEALTH RELEVANCE: This is a proposal to study the hypermutation of antibody genes. This process is beneficial, because it can result in highly specific and efficient antibodies against pathogens and cancer. However, it is also dangerous, because it can cause cancer of lymphocytes and autoimmunity.